DE102020100180A1 - Immunochemical device and lateral flow immunoassay method for the determination of pharmaceutical residues and impurities in breast milk - Google Patents

Abstract

Sampling pad for a lateral flow immunoassay for the detection of a concentration of a drug in breast milk, comprising two inert filter layers and a graphene oxide layer, the graphene oxide layer being stacked between the two inert filter layers, and a lateral flow immunoassay.

Description

TERRITORY AND BACKGROUND

The present invention relates to lateral flow immunoassays (LFIA) for the detection of pharmaceuticals in breast milk. It also relates to the fat and protein depletion of milk samples that are subjected to drug residue detection.

Lateral flow immunoassays (also known as dipstick assays) are used in pregnancy tests, drug tests (on the street), and beverage testing. The basic substances used in these applications are aqueous. In contrast to the basic substances that are usually analyzed, breast milk is rich in fat and proteins. Conventionally configured lateral flow immunoassays are therefore not applicable.

SHORT SUMMARY

According to the proposed method, a breast milk sample is taken with a polymer sponge previously treated with buffer, the taken sample is pretreated by filtration through a graphene oxide layer and the analyte is through a competitive LFIA, comprising antibody-coated gold nanoparticles as a marker on a nitrocellulose strip, which is a plasma treatment and a Has undergone non-contact spotting in the control and test zones. According to the invention, a sample pretreatment step was added to the assay. An integrated sample pad comprising a graphene oxide layer is proposed. Physiological fluids such as breast milk, blood plasma, serum, saliva or urine can be applied directly to the cartridge. The signal reading is carried out with a hand-held reader or a smartphone with a camera and a barcode reader app. A lower limit of detection for diclofenac (as an exemplary analyte) in breast milk of 0.08 µg / l and a recovery of about 92% have been proven. The proposed assay is simple, sensitive, fast and inexpensive.

Figure list

• The 1-3 are consumables used for sample collection and filtration.
• 4th represents different breast milk samples with different fat content.
• The 5 and 6th represent breast milk filtrates obtained on different types of filter materials:
• 5A represents breast milk before and after filtration through a filter with an integrated graphene oxide layer,
• 5B represents breast milk after filtration through the filter analog without the graphene oxide layer,
• 5C represents breast milk with high fat and protein levels after filtration through the filter with an integrated graphene oxide layer,
• 5D represents breast milk with low fat and protein levels after filtration through a filter with an integrated graphene oxide layer.
• 6th shows breast milk with different fat and protein levels after filtration through the filter with an integrated graphene oxide layer. The graph in
• 7th shows the UV-visible absorption spectrum of different types of breast milk samples after filtration with the proposed filter based on graphene oxide (S1 = sample 1R2 (low fat level), S2 = sample 1R (high fat level), S3 = sample 1R (high fat level) in dilution 1: 50, S4 = sample 1R2 (low fat level) in a 1:50 dilution).
• 8A and 8B represent one embodiment of a lateral flow immunoassay device consisting of a test strip and a sample collector.
• The 9-15 are chromatograms obtained by LC-MS / MS (liquid chromatography-tandem mass spectroscopy), which indicate the presence of diclofenac (DCF) in all (spiked) samples ( 9 = Sample 1R1, 10 = Sample 4.1R1, 11 = Sample 2R1, 12th = Sample 1R2, 13th = Sample 4.2R1, 14th = Sample 4.2R2, 15th = Sample 61R1). The samples were filtered through a filter as suggested filtered with an integrated graphene oxide layer, then diluted in Milli-Q water to adjust to the relevant determination range of the LC-MS / MS method.
• 16a Fig. 10 illustrates a top view of the test strip and FIG 16b a sectional view of the test strip.
• 17th illustrates schematically the formation of graphene oxide from graphite by oxidation.
• 18a represents the proposed structure of graphene oxide (GO).
• 18b represents the proposed structure of the graphene oxide (GO) layer.
• 18c is a photograph of graphene oxide "paper".
• 18d is a photo of a filter with an integrated layer of graphene oxide "paper".
• 18th .1a is a photo of graphene oxide powder.
• 18th .1b is a photograph illustrating a filter with a filter based on an integrated graphene oxide powder between filter layers in the sample pad.
• 19th represents photographs of the nitrocellulose (NC) membrane before plasma treatment.
• The 19I and 19II are microscopic photographs of spots obtained by non-contact spotting using a sciFLEXARRAYER S3 spotter (Scienion, Berlin, Germany) to generate the test and control line,
• in the 19III are shown. Here protein molecules migrated laterally in the membrane and are concentrated in a large area. Accordingly, the image of the spots and lines is blurred.
• 20th represents photographs of the NC membrane after plasma treatment.
• 20I and 20II are microscopic photographs of spots obtained by non-contact spotting using the sciFLEXARRAYER S3 spotter to generate the test and control line,
• in the 20III are shown. The plasma treatment increased the binding capacity of the membrane and the proteins are concentrated in a narrower area, which results in very sharp lines.
• 21 (Inset) represents a photo of LFIA strips (gold nanoparticles as markers) obtained with different concentrations of diclofenac (DCF).
• 21 (Graph) represents the interpolated calibration curve of the assay, the signals being read with the Chembio Diagnostics LFIA reader. The curve results from the plot intensity of the T line against the logarithmic value of the DCF concentration. The error bars are standard deviations from three independent measurements.
• 22nd (Inset) represents a photograph of LFIA strips (latex particles as markings) obtained with different concentrations of diclofenac (DCF).
• 22nd (Graph) represents the interpolated calibration curve of the assay, the signals being read with the Chembio Diagnostics LFIA reader. The curve results from the plot intensity of the T line against the logarithmic value of the DCF concentration. The error bars are standard deviations from three independent measurements.
• 23 represents an embodiment of a multiplex LFIA strip in a cassette comprising three T-lines for different antibiotics (classes: macrolides, fluoroquinolones, penicillins).
• 24 represents components of a kit for performing an LFIA for the analysis of pharmaceutical residues in breast milk. It comprises a polymer sponge on a handle for taking breast milk samples, a filter with an integrated graphene oxide layer together with a plunger, which are necessary for the preparation of the layer, and a sample vial to remove the filtrate. Another component is the test strip in its housing (cassette), which is shown here as: double cassette for the parallel analysis of 2 samples for 1 compound or 1 sample for 2 compounds (left); Cassette with 3 T-zones to allow the analysis of 1 sample for 3 compounds (right).

In particular, represents 1 represents a polymer sponge, as it is used for breast milk sampling. The top photograph depicts the sponge, as from https://www.porex.com/en-US/markets/ in-vitro-diagnostics / sample-collection # SalivaUrineCollection available. The diagram below shows the size of the sponge and its handle.

2 provides a disposable sample vial for removing the filtrate (left) (https://www.porex.com/en-US/markets/in-vitro-diagnostics/sample-preparation), a filter insert of a syringe as prepared (middle) and the plunger the syringe used to push the filter sheets into the laboratory-made filter holder.

3 represents a filter arrangement as prepared according to Example 4.

4th represents a series of six different breast milk samples that illustrate a different fat content in breast milk.

5 represents filtrates obtained after filtration of breast milk. In particular, represents 5A a sample before (above) and after (below) filtration through a graphene oxide layer. 5B represents a sample filtered through the glass microfiber filter sheets without graphene oxide. 5C represents a sample after filtration through the filter of Example 3 (polycarbonate nucleopore filter). The fat and / or protein load is still too high for LFIA. 5D represents a sample after filtration through single-layer graphene oxide flakes according to Example 4.

6th represents 5 filtrates from breast milk samples with different fat content after filtration through the graphene oxide filter film according to Example 2. Two milliliters of breast milk were passed through fresh filter layer arrangements as described. The volume withdrawn varies from 200 µl to 1 ml, depending on the original fat content.

7th shows UV-visible spectra of different samples (S 1 = sample 1R2, S2 = sample 1R, S3 = sample 1R in dilution 1:50, S4 = sample 1R2 in dilution 1:50), which still show a high protein content (such as indicated by an absorption band at a wavelength of 280 nm).

8A shows the dropwise application of the filtrate to the LFIA sample port. Usually 1 drop, corresponding to about 50 µl of the sample, was sufficient to perform the assay. 8B illustrates the recordings and design of the lateral flow immunoassay device with positive (left) and negative (right) results.

The table below presents details of the samples that 9-15 are chromatograms obtained by LC-MS / MS (liquid chromatography-tandem mass spectroscopy) which indicate the presence of diclofenac (DCF) in all (spiked) samples. 9 is the chromatogram obtained for sample 1R1, diluted 1:10, according to the table below. 10 corresponds to sample 4.1R1, 1:50; 11 corresponds to sample 2R1, 1:10; 12th corresponds to sample 1R2, 1:20; 13th corresponds to sample 4.2R1, 1:10; 14th corresponds to sample 4.2R2, 1:20; and 15th corresponds to sample 6.1R1, 1: 2.

Fig. No. Sample designation Fat level in the sample Sample dilution DCF according to LFIA [µg / l] DCF according to LC / MS [µg / l] Recovery [%] 9 1R1 high 1: 10 7.6 11 144 11 2R1 high 1: 10 5.3 41 77 12th 1R2 low 1:20 5.6 5.1 90 10 4.1R1 low 1:50 73 71 97 13th 4.2R1 low 1: 10 5.4 4.5 83 14th 4.2R2 low 1:20 7.5 7.3 97 15th 61R1 low 1: 2 3.6 3.2 87

DETAILED DESCRIPTION OF THE INVENTION

According to one embodiment, a sample pad for a lateral flow immunoassay (LFIA) is proposed. The sample pad is used to separate a drug residue from the main components of the basic substance in a body fluid such as breast milk, blood, plasma, saliva and urine adjusted. It includes two inert filter layers and a graphene oxide layer stacked between the two inert filter layers.

The resulting filter stack advantageously allows the fat and / or protein load of a sample to be safely removed without losing the analyte. Therefore, the sample pad enables even an untrained user to take a freshly received sample e.g. B. safely apply a body fluid to an LFIA device without sample pretreatment.

According to one embodiment, the two inert layers comprise a microfiber glass comprising pores with a size of 0.5-2 μm, in particular 1.0-1.2 μm.

Due to their inertia and good flow properties (bubble point), glass microfibers are widely used for filtration purposes.

According to one embodiment, the graphene oxide layer is selected from the list consisting of: a graphene oxide paper consisting of a plurality of graphene oxide nanolayers, and a graphene oxide powder comprising flakes consisting of a plurality of graphene oxide nanolayers.

According to one embodiment, graphene oxide has proven to be a good absorbent for fats and proteins (albumin and immunoglobulins), which z. B. are present in breast milk, without changing the concentration of the model drug diclofenac (DCF).

According to one embodiment, a use of a sample cushion comprising a graphene oxide layer is proposed for the treatment of a sample comprising a physiological fluid immediately before the analysis by removing fat and / or protein from the sample, the physiological fluid being selected from the list consisting of: breast milk, plasma, serum, saliva, urine and whole blood.

According to one embodiment, a volume of the sample is 10-100 μl, preferably 25-75 μl, in particular about 50 μl (ie one drop), with a fat load of the LFIA sample of up to 10%, typically 4.4%, the detection an analyte, e.g. B. a drug such as DCF, in the sample, e.g. B. breast milk, not affected.

Advantageously, up to 250 µl of the original sample can be placed on the suggested sample pad.

According to one embodiment, the thickness of one graphene oxide layer is between 15-20 μm according to AFM (graphene oxide nano-layers) and a graphene oxide powder comprises flakes which consist of several graphene oxide nano-layers between 0.5-0.8 mm.

Advantageously, such a thin layer does not delay the steady flow of liquid along the LFIA strip, but effectively removes associated fat and protein from the applied sample.

According to one embodiment, a lateral flow immunoassay for the detection of an analyte is proposed, the analyte comprising a drug and / or a drug residue in a physiological fluid and the lateral flow immunoassay comprising the filtration of the sample through a graphene oxide layer, the graphene oxide layer is arranged with the sample pad of a lateral flow immunoassay strip.

Such an LFIA advantageously allows a simple detection of analytes in physiological fluids by untrained personnel, in particular by a layperson, “on site” (at the bedside, on the street).

According to one embodiment, the graphene oxide layer is selected from the list consisting of: a graphene oxide paper consisting of a plurality of graphene oxide nano-layers, and a graphene oxide powder comprising flakes consisting of a plurality of graphene oxide nano-layers.

These materials are advantageously commercially available or can be easily prepared, e.g. from graphite by oxidation (cf. 17th ).

According to one embodiment, the medicament and the medicament residue are selected from the list consisting of: carbamazepine (CBZ), caffeine (CAF), cholesterol, diclofenac (DCF), isolithocholic acid (ILA), creatinine, cotinine, atenolol, bisoprolol, metoprolol, carvedilol , Celiprolol, esmolol, labetalol, levobunolol, metipranolol, diazepam, dihydrocodeine, doxepin, ibuprofen, methadone, metoprolol, morphine, diazepam, oxazepam, primidone, gentamicin, sotalol and tonalide, cocaine, benzoylecgonine, herbicide, (MD) , Metolachlor), pesticides (carbofuran, carbaryl), imidacloprid, fenitrothion, clenbuterol, a phthalate ester, a poly- or perfluorinated alkyl substance (PFAS), a bisphenol, an antibiotic penicillin analog, sulfonamide, tetracycline analog, a macrolidone analog, fluoroquinolidone analog Alkaloid and others (e.g. flfenicol, chloramphenicol, lincomycin).

According to one embodiment, a lateral flow immunoassay device is proposed which comprises a sample pad, the sample pad comprising a graphene oxide layer.

Advantageously, the device comprises a housing which has an underside and an upper cover, wherein the upper cover has at least two openings which are arranged over a thin layer or over a membrane which is designed as a strip, i. H. as a membrane strip.

According to one embodiment, the sample pad is attached to a housing cover on a membrane strip made of fiberglass, rayon, polyester, nylon, cellulose, spun polyethylene or other suitable materials with a size of 5-8 mm x 18-20 mm and a thickness of 1- 3 mm, preferably 1.5-2 mm, pressed (cf. 16b) . The sample pad is where the sample is applied (added), which then flows to the NC membrane and is analyzed by flowing along it to the suction pad.

The housing of the device comprising the cover and the underside is advantageously stapled or glued together. Therefore, a rim enclosing a first opening in the lid can be permanently pressed against a membrane stack placed underneath between the lid and the underside of the housing in order to establish fluid contact between the different layers, i. H. the lowermost membrane strip and the layered filter layers, comprising a graphene oxide layer, placed thereon. The size of the sample pad comprising the graphene oxide is selected to accommodate enough sample within the pores of the membrane stack for drug detection by LFIA.

A preferred embodiment of an LFIA device is shown in FIG 8B illustrated. The device includes a housing and a cover ( 8A) having a variable sized inlet port extending from the outer surface of the cover to the interior of the housing for receiving a sample containing the selected one or more analytes to be determined. The inlet port allows the sample to be introduced into a sample collection device attached to the inner surface of the cover as shown in FIG 8A shown is attached. The sample pad can be formed from cotton, fiberglass, rayon, polyester, nylon, cellulose, spun polyethylene, or other suitable materials.

According to one embodiment, the lateral flow immunoassay is designed as a multiplex assay. Accordingly, in addition to the control line (C line), the nitrocellulose strip comprises at least two, preferably three, even more preferably even four different test lines (T lines), each T line comprising a different antigen. Typically, the different T-lines are arranged one after the other along the strip (i.e. in the direction of flow of the liquid in the strip).

Advantageously, by using such a configuration, groups of different analytes can be detected inexpensively, e.g. B. when using IgG-containing polyclonal antisera with an overall broad specificity, which allows a multiplex lateral flow immunoassay to be carried out. Remarkably, the graphene oxide used for sample filtration did not change the concentration of antibiotics as diverse as macrolides, fluoroquinolones and penicillins (cf. 23 ).

• - Entnehmen einer Probe von typischerweise bis zu 2 ml mit einem Polymerschwamm durch Tränken des Polymerschwamms mit der Körperflüssigkeit, wobei der Polymerschwamm zuvor in einer 25 mM Boratpufferlösung behandelt worden war, wobei die Pufferlösung 0,01 M EDTA, 0,05 % Triton X-100, 0,01 % NaN₃ und 0,05 % Tween 20 umfasst, und getrocknet worden war;
• - Filtrieren der entnommenen Probe durch eine Graphenoxidschicht, die entweder als „Graphene Oxide Film - Super Paper“ (www.acsmaterial.com/graphene-oxide-film-super-paper-1061. html) oder als „Single Layer Graphene Oxide Flake“, H-Verfahren (www.acsmaterial.com/single-layer-graphene-oxide-h-method-1001.html oder www.cheaptubes.com/product/single-layer-graphene-oxide-1-20um/) zwischen zwei Glasmikrofaserfiltern (z. B. 1,2 µm Porengröße, Whatman 1822-100, auf der Oberseite und 1,0 µm Porengröße, Whatman 1821-100, auf der Unterseite eines Filter-Sandwiches) bereitgestellt ist; wobei eine Dicke des Graphenoxids 15-20 µm zum Erhalten eines Filtrats beträgt, wobei das Graphenoxidpulver Flocken umfasst, die aus mehreren Graphenoxidnanolagen einer lateralen Größe zwischen 0,5-0,8 mm bestehen;
• - Durchführen eines kompetitiven Lateral-Flow-Immunoassays mit etwa 50 µl (typischerweise 1 Tropfen) des Filtrats wie im vorherigen Schritt erhalten; und
• - Bestimmen einer Menge des Medikamentenrückstands in der Probe unter Verwendung einer Eichkurve.

According to another embodiment, a method for the detection of medicament residues in a body fluid is proposed. The procedure consists of the following steps:

• - Taking a sample of typically up to 2 ml with a polymer sponge by soaking the polymer sponge with the body fluid, the polymer sponge having previously been treated in a 25 mM borate buffer solution, the buffer solution being 0.01 M EDTA, 0.05% Triton X- 100, 0.01% NaN ₃ and 0.05% Tween 20 and dried;
• - Filtering the sample taken through a graphene oxide layer, which is either available as "Graphene Oxide Film - Super Paper" (www.acsmaterial.com/graphene-oxide-film-super-paper-1061. Html) or as a "Single Layer Graphene Oxide Flake", H-method (www.acsmaterial.com/single-layer-graphene-oxide-h-method-1001.html or www.cheaptubes.com/product/single-layer-graphene-oxide-1-20um/) between two glass microfiber filters (e.g., 1.2 µm pore size, Whatman 1822-100, on top and 1.0 µm pore size, Whatman 1821-100, on the bottom of a filter sandwich); wherein a thickness of the graphene oxide is 15-20 µm for obtaining a filtrate, wherein the graphene oxide powder comprises flakes consisting of a plurality of graphene oxide nanolayers of a lateral size between 0.5-0.8 mm;
• - Carrying out a competitive lateral flow immunoassay with about 50 µl (typically 1 drop) of the filtrate as obtained in the previous step; and
• - Determine an amount of drug residue in the sample using a calibration curve.

According to one embodiment, the sponge is soaked with the body fluid typically for 1-5 minutes during the uptake step.

The short time advantageously allows simple handling by a layperson without any additional equipment (e.g. clock or timer) and rapid processing of the test.

According to one embodiment, the body fluid is breast milk and the drug, the residual concentration of which is detected, is selected from diclofenac and an antibiotic, the antibiotic being selected from the classes of macrolides, fluoroquinolones and penicillins.

The adverse effects of diclofenac and antibiotics on infants are well known. Therefore, preventing intoxication is extremely desirable.

The terms “graphene”, “graphene oxide”, “graphene oxide nano-layer” and “graphene oxide” describe two-dimensional carbon structures and are used synonymously throughout the present patent specification.

Unless clearly stated to the contrary, each embodiment described above can be combined with any other embodiment or embodiments.

A full and enabling disclosure of the present invention, at its best mode, to those skilled in the art is set forth in the remainder of the specification, including reference to the accompanying figures, which form a part hereof, and in which we refer to specific embodiments and features of the invention by way of illustration represent. It is to be assumed that other embodiments can be applied and structural or logical changes can be made without departing from the scope of the present invention. The following detailed description is therefore not to be taken in a limiting sense, and the scope of the present invention is defined by the appended claims.

Methods used for the determination of pharmaceutical compounds (drugs) in breast milk are used in research laboratories or specialized clinical laboratories. A well established instrumental method is high performance liquid chromatography (HPLC), especially in combination with mass spectroscopy (HPLC-MS). However, their main disadvantage is the need for expensive equipment and trained personnel. The method is not sufficiently sensitive and robust and therefore requires intensive sample preparation, which makes it expensive and time-consuming. It is therefore unsuitable for screening a large number of samples and entirely unsuitable for use in a domestic setting by a layperson, in a medical examination room or in a pharmacist's laboratory.

LFIA, d. H. Pregnancy test-like assays, because of their straightforward method, low cost, naked eye evaluation, ease of use, and small size, are very valuable as assay systems for the home, bedside, or on the street. However, since milk, and especially breast milk, is a highly complex basic substance with significant amounts of lipids and proteins, the determination of residual traces of pharmaceuticals and their metabolites requires extensive sample extraction and preparation before analysis. This prevents their analysis by a layperson, especially when a more precise value (and not just a yes / no answer) is to be obtained.

In LFIA, a liquid sample containing the analyte in question or its extract moves along a strip of a membrane and thereby passes through various zones in which molecules ("antigens") have been attached that have more or less specific interactions with the analyte. Therefore, a typical LFIA format includes a surface layer to transport the sample from the sample pad via the conjugate release pad along the strip hitting the detection zone to the absorbent pad. Current membrane strips often include nitrocellulose, but can also be made from nylon, polyethersulfone, polyethylene, or even quartz glass.

The technical aim of the described embodiments is therefore to provide an effective method for sample pretreatment, in particular for fat and protein depletion of breast milk samples. Another aim is to provide reliable selection criteria for a membrane material suitable as an LFIA strip and a method for plasma pretreatment, in particular of nitrocellulose. Furthermore, it is an object of the invention to provide a system comprising an LFIA kit consisting of a filter adapted to a pipette for receiving a fixed amount of the filtered sample and a plastic housing for the strip, the housing carries a QR code. The QR code is adapted to direct a user to a webpage with a download link for a smartphone app, where the app reads the test lines and sends the quantitative results back to the user for interpretation, resulting in a reproducible readout of LFIA- Results allowed by exactly this smartphone.

A bar code reader adapted to read a bar code on the test / test package is used to retrieve calibration curve data present in the portal. A user login is suggested for the traceability of results. Users need to log into the app and their results will be linked to their own account after logging in.

According to one embodiment, the nitrocellulose strip can comprise up to 4 different test lines (T-lines), ie it can be designed as a multiplex assay. Therefore, up to 4 different tests can advantageously be carried out and recorded at the same time. For example, using a single strip, four antibiotics from different groups (e.g. macrolides, fluoroquinolones and penicillins) can be detected in the sample at the same time. Therefore, for example, 3 compounds (azithromycin conjugate, ciprofloxacin conjugate and amoxicillin conjugate) were identified on the NC membrane as test lines by means of spotting on a strip (see 23 ) and a goat anti-rabbit immunoglobulin was applied as a control line. Rabbit anti-macrolide IgG, rabbit anti-fluoroquinolone IgG, rabbit anti-penicillin IgG were labeled with gold nanoparticles or latex particles and placed on the conjugate pad. Advantageously, not only a specific analyte (e.g. an antibiotic), but entire groups of antibiotics can be detected simultaneously from a single sample if IgG-containing polyclonal sera that replace a group selectivity, e.g. the macrolide group, are used.

A prescribed time (e.g. 10 minutes) that must pass before users can record a test result (a skip feature available during development) is included to aid ease of use and the likelihood of false-negatives and false - reduce positive results. Reading the results using the smartphone app helps to get correct diagnostic values in a home setting.

The app (ie the mobile application, also referred to as a mobile app, is a computer program or software application that is designed to run on a mobile device such as a phone, tablet or a smart watch) contains a database of drugs and nutritional supplements that can affect breastfeeding. It contains information about the levels of such substances in breast milk and the infant's blood and possible adverse effects on the breastfed infant. The app helps to track the breastfeeding process. It can also contain accompanying information with photos and video clips, basic information about and requirements for breastfeeding (how to breastfeed, how is breast milk taken and how it is stored, nutrition during breastfeeding, etc.). Scanning the QR code also allows the database administrator to successfully track website and app activity using suitable software tools.

The selection and modification of the membranes in the absorbent pad and the membranes of the strips in the lateral flow device have been optimized in such a way that they can cope with the mother's milk base. The viscosity of breast milk is an important property that greatly influences the flow rate. As we have observed, membranes with a slower flow rate can discriminate the difference in fluid viscosity more effectively than membranes with a faster flow rate. The membranes produced the following capillary rise times for one 4 cm separation distance along the membrane: CNPC 8 µm: 220 sec, CNPC 12 µm: 110 sec, CNPH 150 µm: 90-150 sec from MDI (www.mdimembrane.com). For a separation distance of 12 mm on the LFIA device, the time difference between separation times for breast milk and deionized water is about 5 seconds on CNPC 8 µm, 8 seconds on CNPC 12 µm and 13 seconds on 150 CNPH. A CNPC 8 µm membrane is too slow. The CNPH 150 μm type without plasma treatment gave a large uptake for the T line (BSA conjugate was applied to the test line by means of spotting, cf. 19th ), because the amount of detector particles is concentrated in a large area. A suitable NC membrane is CNPC 12 µm in combination with an absorbent pad comprising AP 110 (MDI), which leads to an increase in the flow rate of 15 seconds for the entire length of the strip. In addition, this made it possible to increase the sensitivity of the assay, which allows more reliable results. Nitrocellulose (NC) membranes bind proteins electrostatically through the interaction of the strong dipole of the nitrate ester with the strong dipole of the peptide bonds of the protein.

A plasma treatment in ammonia and ammonia-hydrogen mixtures for a CNPC-12 µm membrane ( 19th ) are used to modify the surface of nitrocellulose membranes. The plasma treatment was found to be effective in increasing the binding capacity of the NC membrane by increasing the total amount of grafted amino groups, since H atoms produced in the plasma discharge were able to convert grafted nitro groups into amino groups through a reduction process.

EXAMPLES

Example 1 - Sample Collection Sponge Preparation

A polyester sponge like in 1 was prepared with a 25 mM sodium borate buffer solution, pH 8.5 (prepared by adding HCl), comprising 0.01 M EDTA, 0.05% Triton X-100, modified. The sponge was immersed in the indicated solution for 2 minutes. The sponge was then dried overnight at room temperature in a desiccator over silica gel or calcium sulfate (Drierite ™, Sigma-Aldrich) and stored in a closed box at room temperature until use.

Example 2 - Preparation of the sample pretreatment filter A

Filters for sample pretreatment, ie cell, protein and fat removal, were prepared from the uppermost part of two-part 10 ml Luer slip syringes (BD Difco ™ Discardit ™) by cutting off their front end with a sharp knife (cf. 3 ). A circular piece of glass microfiber filter with a pore size of 0.1 µm (Whatman 1821-100) of an appropriate size was placed on the underside of the front end and squeezed with the plunger of the syringe. Then a layer of “Graphene Oxide (GO) Film - Super Paper” (www.acsmaterial.com/graphene-oxide-filmsuper-paper-1061.html) was pressed against the first layer. Another glass microfiber filter with a pore size of 1.2 µm (Whatman 1822-100) of a suitable size was pressed firmly onto the GO layer with the plunger.

Example 3 - Preparation of the sample pretreatment filter B

Unlike in Example 2, instead of the graphene oxide film, a polycarbonate track-etch membrane (Whatman® Nuclepore ™) with a pore size of 12.0 μm was used between the two glass microfiber filters with a pore size of 1.0 μm and 1.2 μm.

Example 4 - Preparation of the sample pretreatment filter C

Unlike in Example 2, instead of the graphene oxide film, a layer of about 0.5-0.8 mm “Single Layer Graphene Oxide Flake”, H method (www.acsmaterial.com/single-layer-graphene-oxide-h-method -1001.html) is used between the two glass microfiber filters with a pore size of 1.0 µm and 1.2 µm.

Example 6 - Filtration of Diclofenac Spiked Breast Milk Samples

According to a practical example 5, a fat and protein depletion was carried out according to the table below by filtration of original breast milk samples which had been spiked with diclofenac. The samples were gravimetrically spiked with different DCF concentrations (see table). The samples were stored at -20 ° C until their analysis or stored at 4 ° C until their analysis within 3 days. Sample no. Sample designation Fat level in the sample Sample dilution DCF according to LFIA [µ / l] DCF according to LC / MS [µ / l] Recovery [%] 1 1R high 1: 10 7.6 11 144 2 2R high 1: 10 5.3 41 77 3 1R low 1:20 5.6 5.1 90 4th 4.1R1 low 1:50 73 71 97 5 4.2R1 low 1: 10 5.4 4.5 83 6th 4.2R2 low 1:20 7.5 7.2 97 7th 61R1 low 1: 2 3.6 3.2 87

A woman's diet can affect the composition of her breast milk; the composition changes dynamically within a feeding process, with the time of day, during breastfeeding and between mothers and populations. Normally, mature human breast milk contains 3% -5% fat, 0.8% -0.9% protein, 6.9% -7.2% carbohydrates, expressed as lactose, and 0.2% minerals, expressed as ash. Their energy content is 60-75 kcal / 100 ml. The fat level in breast milk is determined using the Röse-Gottlieb method (International Dairy Federation. 1987. Milk. Determination of fat content - Röse Gottlieb gravimetric method, IDF, Brussels, Belgium) . In this study, a low fat level is ≤ 2% and a high level is ≥ 5%.

The samples were spiked with different DCF concentrations and then diluted in Milli-Q water to adjust to the relevant determination range of the LC-MS / MS method. Dilution of the sample up to 1:50 did not affect the recovery. Rather, a lower dilution (1: 2) seems to worsen the recovery for DCF concentrations, perhaps due to the limit of quantification (LOD) of LC-MS / MS, which is 5-10 µg / l (see sample no. 7 with a DCF Concentration below 5 µg / l). If breast milk has a high fat level (but <10% fat), it is recommended to dilute the sample for the LC-MS / MS method at least 1: 2 in order to avoid contamination of the chromatographic system.

The primary mouse anti-DCF IgG monoclonal antibodies were kindly provided by Prof. Dr. Dietmar Knopp (Technical University of Munich) provided. 40 nm gold nanoparticles (BioReady ™ 40 nm Carboxyl Gold, product number AUXR40) were used as marking. They were obtained from NanoComposix (https://nanocomposix.com/). These GNP are functionalized with a tightly bound monolayer containing terminal functional carboxylic acid groups that can be activated by EDC / NHS chemistry. Carboxy surfaces have been used to covalently attach free amino groups from antibodies to the surface of the particles. The marker used was carboxyl-modified colored microspheres (Estapor®) from Millipore (product number K1-030). Latex-based microspheres with a size of 0.3 µm were also used. The antibodies were covalently attached to the surface of carboxylated latex using EDC / NHS chemistry.

The nitrocellulose (NC) membrane used was laminate type 150 CNPH from MDI (www.mdimembrane.com) treated in a plasma. Their characteristic parameters are as follows:

PHYSICAL TESTS

Polyester thickness (µm): 102 Membrane thickness (µm): 103

FUNCTIONAL TESTS

Wicking rate, 4 cm distance (seconds) (blocked): 122 Wicking rate, 4 cm distance (seconds) (normal saline solution): 145 Point distribution for 10 µl solution (seconds): 3-5 Adhesion test (24 hours in H ₂ O): passed test

DIMENSION TESTS

Membrane width (mm): 25th Dimensions of the PVC (mm): 58 × 260 Color of PVC: White Thickness of PVC (µm): 250

The material for the carrier layer on which different parts of the lateral flow immunoassay (NC membrane, absorbent pad and sample pad) are installed (using a suitable adhesive) was PVC (polyvinyl chloride).

The plasma treatment equipment for the NC membrane mainly consists of a glass reactor with a non-symmetrical electrode configuration. The low frequency power (50-75 kHz) provided by an industrial generator is capacitively coupled to the plate-type electrode. The NC membrane with the PVC carrier layer (58 x 260 mm) is placed on the grounded cylinder. The real time exposure of the polymer is calculated from the total duration of the plasma discharge and the plasma width on the membrane. For a total plasma treatment time of 1 s, the calculated real time is 0.03 s. The gas is introduced through mass flow controllers (MKS Instruments, Inc.) and the pressure is monitored with an MKS capacitive meter.

The general structure of the LFIA strips used and the geometric properties of the cushions used in the LFIA are shown in FIG 16 shown.

Practical results obtained using the described LFIA for DCF detection in breast milk are given in the table below. Sample no. Sample designation Fat level in the sample LC / MS [µg / l] LFIA [µg / l] Recovery LFIA [%] 1 1R high 11 12th 111 2 2R high 41 49 118 3 1R low 5.1 4.8 94 4th 4.1R1 low 71 82 116 5 4.2R1 low 4.5 4.2 93 6th 4.2R2 low 7.2 7.0 97 7th 61R1 low 3.1 2.9 93

1. 1. Wir beanspruchen ein Verfahren zur Analyse von Muttermilch von einem Subjekt durch einen Laien;
2. 2. Wir beanspruchen, dass pharmazeutische Rückstände und Verunreinigungen in einer Brustmilchprobe sensitiv bestimmt werden können;
3. 3. Wir beanspruchen eine technische Maßnahme, mit der die Probe behandelt wird, um die Freisetzung des Analyten von einem Bindungsprotein oder einem Bindungsagens zu vereinfachen;
4. 4. Wir beanspruchen, dass Graphenoxid verwendet wird, um die Antigenbindung an in dem LFIA-Streifen verwendete Partikel zu reduzieren;
5. 5. Wir beanspruchen eine Plasmabehandlung der Nitrocellulosemembran, die einen gleichmäßigen Probenstrom auf dem Nitrocellulosesubstrat bereitstellt;
6. 6. Wir beanspruchen die Verwendung eines auf der Gehäuseoberseite oder -unterseite aufgedruckten oder angebrachten QR-Strichcodes, der dem Teststreifen einen einzigartigen Code zur Verwendung zuweist und den Download der Smartphone-App zum Lesen und zur Ergebnisauswertung und -übertragung an einen Hosting-Service initiiert;
7. 7. Wir beanspruchen eine Gehäuseoberseite, auf der zwei oder mehr erweiterte Vertiefungen zur Verarbeitung mehrerer Proben untergebracht sind;
8. 8. Wir beanspruchen, dass das Subjekt, von dem die Muttermilch stammt, ein Mensch oder ein beliebiges anderes laktierendes Säugetier ist;
9. 9. Wir beanspruchen als Träger für die Antikörper einen Goldnanopartikel, doch es kann auch ein Quantenpunkt, ein Kern/Hülle-Polymer oder ein Polymer/Kieselerde-Partikel oder ein metallorganischer Gerüstpartikel sein;
10. 10. Wir beanspruchen die Verwendung von Antikörpern, die den Analyten (Pharmazeutika- oder Schadstoffmolekül) binden;
11. 11. Wir beanspruchen eine Lateral-Flow-Testvorrichtung zur Verwendung in Kombination mit einem Probensammler für Muttermilch;

In summary, some aspects of the embodiments described herein can be briefly described as follows:

1. 1. We claim a method of analysis of breast milk from a subject by a layperson;
2. 2. We claim that pharmaceutical residues and impurities can be determined sensitively in a breast milk sample;
3. 3. We claim a technical measure by which the sample is treated in order to facilitate the release of the analyte from a binding protein or a binding agent;
4. 4. We claim that graphene oxide is used to reduce antigen binding to particles used in the LFIA strip;
5. 5. We claim a plasma treatment of the nitrocellulose membrane which provides a uniform sample flow on the nitrocellulose substrate;
6. 6. We claim the use of a QR barcode printed or affixed to the top or bottom of the housing, which assigns the test strip a unique code for use and initiates the download of the smartphone app for reading and for result evaluation and transmission to a hosting service ;
7. 7. We claim a top side of the housing which accommodates two or more enlarged wells for processing multiple samples;
8. 8. We claim that the subject from whom the breast milk is obtained is a human being or any other lactating mammal;
9. 9. We claim a gold nanoparticle as a carrier for the antibodies, but it can also be a quantum dot, a core / shell polymer or a polymer / silica particle or an organometallic framework particle;
10. 10. We claim the use of antibodies that bind the analyte (pharmaceutical or pollutant molecule);
11. 11. We claim a lateral flow test device for use in combination with a sample collector for breast milk;

The present invention has been explained with reference to various illustrative embodiments and examples. These embodiments and examples are not intended to limit the scope of the invention, which is defined by the claims and their equivalents. As will be apparent to those skilled in the art, the embodiments described herein can be implemented in various ways without departing from the scope of the invention. Various features, aspects and functions described in the embodiments can be combined with other embodiments.

Claims (15)

Sample pad for a lateral flow assay, especially a lateral flow immunoassay, for the detection of a concentration of a drug in a body fluid, comprising two inert filter layers and a graphene oxide layer, the graphene oxide layer being stacked between the two inert filter layers. Sample pillow after Claim 1 wherein the two inert layers comprise a microfiber glass comprising pores with a size of 0.5-2 μm, in particular 1.0-1.2 μm. Sample pillow after Claim 1 or 2 wherein the graphene oxide layer is selected from: a graphene oxide paper consisting of a plurality of graphene oxide nano-layers, and a graphene oxide powder comprising flakes consisting of a plurality of graphene oxide nano-layers. Use of a sample cushion, comprising a graphene oxide layer for the depletion of fat and / or protein from a sample of a body fluid, the body fluid being selected from: a breast milk, a blood, a plasma, a saliva and a urine. After using the sample pad Claim 4 for applying a sample volume of 10-100 µl, preferably 25-75 µl, in particular about 50 µl, whereby a fat load of the sample of up to 10% does not impair the detection of an analyte in the sample. Use of the sample cushion, the thickness of the graphene oxide layer being between 0.5-0.8 mm. Lateral flow immunoassay for the detection of an analyte comprising a medicament and / or a medicament residue in a physiological fluid, comprising the filtration of the sample through a graphene oxide layer, wherein the graphene oxide layer is arranged with the sample pad of a lateral flow assay strip. Lateral flow immunoassay Claim 7 wherein the graphene oxide layer is selected from: a graphene oxide paper consisting of a plurality of graphene oxide nano-layers, and a graphene oxide powder comprising flakes consisting of a plurality of graphene oxide nano-layers. Lateral flow immunoassay Claim 7 or 8th , wherein the drug and the drug residue are selected from: carbamazepine (CBZ), caffeine (CAF), cholesterol, diclofenac (DCF), isolithocholic acid (ILA), creatinine, cotinine, atenolol, bisoprolol, metoprolol, carvedilol, celiprolol, esmolol, labetalol , Levobunolol, metipranolol, diazepam, dihydrocodeine, doxepin, ibuprofen, methadone, metoprolol, morphine, diazepam, oxazepam, primidone, gentamicin, sotalol and tonalid, cocaine, benzoylecgonine, THC, MDMA, herbicides (glyphosate, atrazine) Carbofuran, carbaryl), imidacloprid, fenitrothion, clenbuterol, a phthalate ester, a poly- or perfluorinated alkyl substance (PFAS), a bisphenol, an antibiotic penicillin analog, sulfonamide, tetracycline analog, fluoroquinolone analog, macrolide analog, e.g. , Chloramphenicol, lincomycin. A lateral flow immunoassay device comprising a sample pad according to any one of Claims 1 - 3 . Lateral flow immunoassay device according to Claim 10 , wherein the sample pad is mechanically pressed by a housing cover onto a membrane strip made of glass fiber, rayon, polyester, nylon, cellulose, spun polyethylene or other fibrous organic material, the sample pad size being 5-8 mm x 18- Measures 20mm and a sample pad thickness is selected to be between 1 and 3mm. Lateral flow immunoassay device according to one of the Claims 10 or 11 , wherein the membrane strip comprises, in addition to a control line (C line), at least one, preferably up to four different test lines (T lines), each T line comprising a different antigen, which allows a multiplex lateral flow immunoassay to be carried out . A method for detecting a residue of a medicament in a body fluid, the method comprising the following steps: taking a sample of the body fluid with a polymer sponge by soaking the polymer sponge with the body fluid, the polymer sponge previously in a 0.01 M EDTA, 0.05 25 mM borate buffer solution comprising% Triton X-100, 0.01% NaN ₃ and 0.05% Tween 20 was treated and dried; - Filtration of the recorded sample through a graphene oxide layer, which comprises either a graphene oxide film (“super paper”) or a single-layer graphene oxide flake, the graphene oxide layer being arranged between two glass microfiber filters; wherein a thickness of the graphene oxide layer is 15-20 μm and the graphene oxide layer comprises graphene oxide nanolayers with a lateral size of 0.5-0.8 mm; - Carrying out a lateral flow immunoassay with about 50 µl of the collected and filtered sample; - Determine an amount of drug residue in the sample using a calibration curve. Procedure according to Claim 13 , wherein in the uptake step typically up to 2 ml of the sample are taken up by soaking for 1-5 min. Procedure according to Claim 13 or 14th wherein the body fluid is breast milk and the medicament is selected from diclofenac and an antibiotic, the antibiotic being selected from a class such as macrolides, fluoroquinolones and penicillins.